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ZOOLOGICAL SCIENCE 18: 505–514 (2001) © 2001 Zoological Society
of Japan
* Corresponding author: FAX. +49-221-9417285.E-mail:
[email protected]
Chromosome Data for Malagasy Poison Frogs (Amphibia: Ranidae:
Mantella) and Their Bearing on
Taxonomy and Phylogeny
Gaetano Odierna1, Miguel Vences2*, Gennaro Aprea1, Stefan
Lötters3
and Franco Andreone4
1Dip. Biologia Evolutiva e Comparata, Università di Napoli
“Federico II”,Via Mezzocannone 8, 80134 Napoli, Italy
2Museum national d’Histoire naturelle, Laboratoire des Reptiles
et Amphibiens,25 rue Cuvier, 75005 Paris, France
3Zoologisches Forschungsinstitut und Museum A. Koenig,
Adenauerallee 160,53113 Bonn, Germany
4Sezione di Zoologia, Museo Regionale di Scienze Naturali,Via G.
Giolitti 36, 10123 Torino, Italy
ABSTRACT—We compared chromosome morphologies for 11 species of
Malagasy poison frogs, genusMantella, and three outgroup taxa
(genus Mantidactylus) using conventional and fluorescence staining
tech-niques. All species studied had a karyotype of 2n=26, with
five larger and eight smaller chromosome pairs.The 11th pair was
acrocentic in Mantella nigricans which represents the first such
observation in the genus.The nucleolus organizer region (NOR) was
located at secondary constrictions on chromosome pair 2 in
allMantella studied and in Mantidactylus grandisonae (while located
on other chromosomes in all other speciesof Mantidactylus studied
so far). Heterochromatin distribution was highly variable among
Mantella species;C-bands positively staining with DAPI and CMA3
were observed. The possible structure of these bands,seemingly
containing both A+T rich and C+G rich heterochromatin, is
discussed. Phylogenetic reconstruc-tion using chromosomal
characters provided very little information. Evolution of the
characters studied isprobably either too fast (heterochromatin
arrangement) or too slow (NOR location) to match the
maincladogenetic events among Mantella species groups.
INTRODUCTION
Madagascar is famous for its organismal diversity andhigh degree
of endemism. Among the most speciose verte-brate clades are the
mantellines. This lineage, includingMantella and Mantidactylus
(Glaw and Vences, 1994; Glawet al., 1998) has been considered as
subfamily Mantellinae ofthe cosmopolitan frog family Ranidae
(Blommers-Schlösser,1993) or as separate family Mantellidae
(Dubois, 1992).Mantellines are characterized by a specialized
mating behav-ior involving absence of a strong mating amplexus
(Blommers-Schlösser, 1993; Glaw et al., 1998). They are a
monophyleticgroup as supported by morphological and molecular
studies(Glaw et al., 1998; Richards and Moore, 1998; Richards
etal., 2000).
Currently more than 65 nominal species of Mantidactylusare known
(Glaw and Vences, 1999, 2000; Glaw et al., 2000).
They are highly diverse, ranging from large and semiaquaticto
minute and scansorial, and from species with fairlygeneralized
tadpoles to highly specialized species withdirect development
(Blommers-Schlösser and Blanc, 1991;Glaw and Vences, 1994).
Molecular data demonstrated thatMantidactylus is not monophyletic:
the molecular study ofRichards et al. (2000) supported
relationships of Mantellato species of Mantidactylus belonging to
the subgeneraBlommersia, Guibemantis, and Pandanusicola.
In contrast, the genus Mantella is a well defined mono-phyletic
unit (Vences et al., 1998a, b) containing about 17morphologically
poorly differentiated species (Vences et al.,1999). Mantella are
attractive, small diurnal frogs which accu-mulate skin alkaloids,
most probably by uptaking arthropodprey (Daly et al., 1996; 1997),
and characterized by apose-matic coloration. Hypotheses of
intrageneric relationships havebeen proposed based on a number of
different character sets(Pintak et al., 1998; Vences et al., 1998b,
c).
The chromosomes of mantelline frogs have beendescribed thus far
mainly by Blommers-Schlösser (1978). She
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G. Odierna et al.506
described general chromosome morphology in 24 species
ofMantidactylus and Mantella aurantiaca, M. betsileo, and
M.haraldmeieri. In addition, Pintak et al. (1998) provided dataon
Mantella aurantiaca, M. baroni, M. betsileo, M. expectata,M.
haraldmeieri, M. laevigata, and M. viridis. The present
studycomplements these earlier contributions by adding new spe-cies
of Mantella to the data set. Besides general chromosomemorphology,
we also studied the distribution and compositionof heterochromatin
and the location of nucleolus organizerregions (NORs). Our goals
were (1) to test recent Mantellaclassification by searching for
chromosomal differencesbetween closely related taxa, and (2) to
obtain a new set ofdata to test hypotheses of Mantella
phylogeny.
MATERIALS AND METHODS
We examined a total of 36 specimens of Mantella belonging to11
different species (see appendix for voucher specimens), whichbelong
to the M. aurantiaca group (M. aurantiaca), M. betsileo group(M.
betsileo, M. cf. betsileo, M. expectata, M. viridis), M. cowani
group(M. baroni, M. cowani, M. nigricans), M. laevigata group (M.
laevigata)and M. madagascariensis group (M. madagascariensis, M.
pulchra)as defined by Vences et al. (1999). Six specimens of three
species ofMantidactylus were used for outgroup comparisons. Voucher
speci-mens have been deposited at the Museo Regionale di
Scienze
Naturali, Torino (MRSN) and the Zoologisches Forschungsinstitut
undMuseum Alexander Koenig, Bonn (ZFMK). Each specimen was
treatedwith 0.01 ml per g body weight of a 0.5 mg/ml colchicine
solution.Four hr later, animals were sacrificed using 0.1%
tricainemetasulfonate (MS-222). Intestines, spleens, lungs, and
gonads wereremoved and incubated for 30 min in a 0.7% sodium
citrate solution.Chromosomes were obtained by air drying and
scraping as describedby Olmo et al. (1986). Besides conventional
staining (5% Giemsa atpH 7), the following techniques were applied:
(1) AgNO3-banding ofNORs following Howell and Black (1980); (2)
staining with the C+Gspecific fluorochrome chromomycin A3 (CMA3)
according to Saharand Latt (1980), with a reduced exposure (a few
seconds) to the nonfluorescent dye, methyl green; (3) the A+T
specific fluorochrome, DPI/distamycin (DA), and CMA3/DA (DAPI:
Schweizer 1976); (4) C-band-ing as described by Sumner (1972),
incubating the slides for 5 min at45°C in Ba(OH)2; (5) in situ
digestion with Alu I endonucleases (com-pare Mezzanotte et al.
1983). Suitable results were achieved by stain-ing, either
separately or sequentially, with CMA3 and DAPI afterhydrolysis in
Ba(OH)2 or digestion with Alu I.
Metaphase chromosomes were stained with Giemsa; AgNO3
andC-banding/Giemsa were viewed on a Zeiss PHOM III phase
contrastmicroscope, whereas the fluorochrome-stained metaphases
(CMA3and DAPI) were viewed on a Leitz epifluorescent microscope. Of
eachtaxon, at least four Giemsa-stained metaphases and two
metaphasesstained with each of the banding methods used were
studied. Imageswere digitized using a scanner. Karyotypes were
constructed usingAdobe Photoshop 3.0. Measurements to determine
relative chromo-some length (rl; percentage ratio between the
length of each chromo-
Table 1. Relative chromosome lengths (rl) of chromosomes 1–13 in
the species studied. Data are mean values with standard
deviations.
Species rl (1) rl (2) rl (3) rl(4) rl (5) rl (6) rl (7) rl (8)
rl (9) rl (10) rl (11) rl (12) rl (13)
Mantella aurantiaca 16.3±0.5 13.0±0.8 12.0±0.3 11.5±0.5 9.9±0.4
7.1±0.3 5.9±0.3 5.5±0.2 5.2±0.3 5.0±0.3 4.7±0.4 4.6±0.3
4.2±0.5Mantella baroni 16.1±0.6 13.3±0.7 12.2±0.8 10.5±0.5 10.2±0.3
6.8±0.2 5.6±0.3 5.6±0.4 5.1±0.3 4.8±0.2 4.3±0.3 4.5±0.4
3.9±0.2Mantella betsileo 16.7±0.8 14.3±0.5 12.1±0.6 10.6±0.6
9.9±0.3 6.3±0.7 5.7±0.4 4.9±0.4 4.8±0.3 4.4±0.4 3.8±0.3 3.6±0.2
3.3±0.4Mantella cf. betsileo 15.3±0.3 13.4±0.3 12.8±0.5 10.8±0.8
9.8±0.1 6.8±0.3 6.6±0.8 4.5±0.3 3.9±0.5 4.4±0.3 3.9±0.1 3.8±0.1
3.4±0.5Mantella cowani 14.2±0.8 12.8±0.5 11.8±0.3 11.3±0.1 10.1±0.4
6.8±0.2 5.8±0.3 5.4±0.3 5.2±0.3 5.4±0.8 4.0±0.4 3.9±0.7
3.8±0.8Mantella expectata 15.7±0.6 13.2±0.7 11.9±0.4 10.8±0.5
9.9±0.4 5.9±0.3 5.7±0.3 5.4±0.2 5.2±0.3 4.4±0.2 4.2±0.3 3.8±0.4
3.7±0.2Mantella laevigata 14.8±0.6 13.3±0.4 11.7±0.3 11.3±0.2
10.1±0.5 6.0±0.5 5.7±0.2 5.6±0.3 5.2±0.3 4.7±0.2 4.3±0.1 3.8±0.4
3.7±0.2Mantella madagascariensis 14.3±1.0 12.1±0.9 11.1±0.7
11.0±0.5 10.0±0.2 6.7±0.2 5.6±0.5 5.5±0.5 5.4±0.7 4.9±0.5 4.4±0.5
4.3±0.1 3.7±0.7Mantella nigricans 16.3±0.1 14.1±0.3 10.8±0.6
11.0±0.1 9.9±0.1 7.1±0.3 6.0±0.5 5.2±0.2 5.3±0.4 4.8±0.3 4.4±0.6
4.2±0.2 3.9±0.4Mantella pulchra 14.3±0.9 12.9±0.7 12.8±0.8 10.8±0.2
10.2±0.3 7.2±0.4 5.4±0.2 5.3±0.1 5.0±0.1 4.6±0.1 4.2±0.5 4.1±0.3
4.0±0.2Mantella viridis 16.4±0.4 13.2±0.4 11.8±0.3 11.2±0.3
10.0±0.4 6.1±0.4 5.6±0.1 5.3±0.2 5.1±0.1 4.6±0.3 3.9±0.4 3.9±0.3
3.5±0.3Mantidactylus grandisonae 18.3±0.6 15.8±0.7 15.3±0.5
12.5±0.5 9.1±0.4 5.7±0.3 5.5±0.4 4.6±0.2 4.1±0.4 3.8±0.3 3.7±0.4
3.3±0.3 3.0±0.2Mantidactylus bicalcaratus 14.5±0.6 13.1±0.4
11.6±0.7 11.4±0.9 9.3±0.4 6.4±0.2 6.0±0.5 5.5±0.3 5.2±0.4 4.9±0.5
4.5±0.6 4.1±0.6 3.8±0.3Mantidactylus cf. punctatus 15.3±0.7
12.9±0.1 12.1±0.3 11.8±0.7 9.8±0.5 7.0±0.3 6.5±0.3 5.4±0.8 5.1±0.3
5.0±0.3 4.6±0.3 4.3±0.5 3.6±0.4
Table 2. Centromer indices (ci) of chromosomes 1–13 in the
species studied. Data are mean values with standard deviations.
Species ci (1) ci (2) ci (3) ci(4) ci (5) ci (6) ci (7) ci (8)
ci (9) ci (10) ci (11) ci (12) ci (13)
Mantella aurantiaca 46.9±2.0 39.6±3.0 32.3±3.3 40.3±2.7 42.5±1.9
30.7±2.3 45.9±4.0 45.3±2.2 44.1±3.7 44.6±2.8 40.0±2.8 44.0±3.5
43.9±3.3Mantella baroni 45.1±2.2 39.5±2.9 31.9±1.6 40.4±2.0
40.7±2.3 30.9±2.7 45.9±3.4 44.9±3.4 42.9±4.0 44.6±2.8 40.0±.8
42.7±2.4 39.9±2.1Mantella betsileo 46.0±2.0 38.9±2.2 30.9±3.3
38.6±1.7 43.8±1.6 45.8±1.2 44.5±5.6 44.0±3.2 42.3±4.0 45.8±2.2
40.3±2.1 42.0±3.3 43.0±3.2Mantella cf. betsileo 45.4±2.3 36.8±3.1
31.5±3.1 39.0±1.1 42.0±3.1 29.9±2.3 45.3±2.7 42.4±3.2 43.0±2.1
46.1±2.1 40.0±1.3 41.2±3.2 42.8±2.7Mantella cowani 46.2±2.8
38.6±2.5 32.9±1.8 40.8±2.0 43.1±1.8 30.0±2.6 43.3±2.9 44.8±2.1
46.1±3.1 45.8±2.1 40.0±2.4 41.1±2.5 43.1±3.3Mantella expectata
45.9±2.1 38.6±4.3 31.7±2.1 39.1±4.1 39.7±1.4 30.2±4.1 44.7±2.0
40.7±2.6 44.0±1.8 41.5±3.5 39.3±2.7 42.6±4.8 42.7±3.2Mantella
laevigata 45.5±3.5 39.4±3.4 32.0±4.4 37.3±3.1 41.7±1.5 33.2±2.8
40.8±4.3 44.4±2.5 43.2±3.9 47.6±2.6 39.5±1.9 43.2±2.9
42.4±4.4Mantella madagascariensis 44.1±3.4 39.1±2.3 31.1±3.7
39.3±2.5 42.9±4.1 29.8±2.9 42.9±2.8 42.4±3.8 44.2±1.9 45.7±2.2
41.2±1.6 42.3±3.4 43.1±2.9Mantella nigricans 44.2±4.1 39.6±2.9
31.8±4.0 38.6±3.1 41.3±3.0 32.2±3.0 44.2±1.5 43.5±3.1 42.0±4.0
46.2±2.2 0.09±1.8 43.1±1.5 42.7±0.2Mantella pulchra 45.3±1.2
38.2±4.0 31.9±1.5 37.3±2.5 41.2±3.1 31.3±2.2 43.4±2.1 44.8±3.1
43.9±4.0 45.2±2.7 40.2±1.7 42.6±1.2 43.0±2.1Mantella viridis
45.7±1.8 38.7±2.7 32.2±4.8 38.6±2.2 41.8±2.1 33.2±1.8 41.6±3.1
45.7±2.4 46.1±2.2 45.1±1.9 42.5±2.9 42.0±3.2 44.1±4.4Mantidactylus
grandisonae 39.0±5.0 33.3±5.3 38.0±4.6 41.2±4.0 41.6±3.8 31.8±3.3
40.9±3.9 45.9±4.0 40.6±3.5 33.3±3.0 42.8±3.7 46.2±4.0
41.7±3.6Mantidactylus bicalcaratus 44.0±2.2 41.1±2.0 31.3±3.8
30.9±3.0 39.9±2.8 34.1±2.5 42.3±4.0 41.9±2.1 39.1±1.9 40.3±1.7
43.2±2.9 40.5±1.5 40.1±1.9Mantidactylus cf. punctatus 43.9±3.2
40.2±2.3 34.0±2.8 30.2±1.6 41.0±2.1 35.0±2.3 43.4±3.6 40.6±2.0
39.1±1.9 40.5±2.0 41.6±3.1 39.7±1.8 41.1±1.2
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Chromosome Data for Malagasy Poison Frogs 507
some to the total length of all the chromosomes) and centromer
index(ci; ratio between short arm and total length of each
chromosome)were carried out using the digitized images. In the
following accounts,DAPI-positive bands are referred to simply as
“DAPI+”, CMA3-posi-tive bands as “CMA+”.
Cladistic analysis was carried out using PAUP*, version 4
beta(Swofford, 1998). We calculated Maximum parsimony and
Neighbor-joining (NJ) trees based on total character differences,
and tested the
trees by running 2000 bootstrap replicates. An interspecific
principalcomponent analysis (PCA) of mean rl and ci values was
performedwith SPSS for Windows, version 6.1.2.
RESULTS
All species of Mantella and Mantidactylus examined had
Fig. 1. Giemsa stained karyotype of Mantella taxa studied: M.
aurantiaca (A), M. baroni (B), M. betsileo (C), M. cf. betsileo
(D), M. cowani (E),M. expectata (F), M. laevigata (G), M.
madagascariensis (H), M. nigricans (I), M. pulchra (J), M. viridis
(K). The AgNO3 stained NOR bearing 2ndchromosome pair is also
reported.
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G. Odierna et al.508
a karyotype of 2n=26 chromosomes with five larger and
eightsmaller chromosome pairs. All chromosomes were metacen-tric
except for the submetacentric third and sixth pair (Table 1and 2;
Fig. 1). The only deviations from this pattern wereexhibited by M.
betsileo which showed a metacentric sixthpair, and M. nigricans in
which the 11th pair was acrocentric(Table 1 and 2). All Mantella
species as well as Mantidactylusgrandisonae had a secondary
constriction near the centromereon the short arm of the 2nd
chromosome pair; this secondaryconstriction was selectively stained
by the AgNO3 and theCMA3/MG staining, indicating that it
corresponds to the NOR.
In Mantidactylus cf. punctatus and M. bicalcaratus, the NORwas
interstitial on the short arm of the 1st chromosome pair(Fig.
1).
Eight principal component factors with an Eigenvalue >10were
obtained by PCA. The first and second principal compo-nent factors
together explained 56.8% of the observed totalvariation. The first
factor was mainly influenced by relativechromosome lengths:
although the highest principal compo-nent loading was that of the
centromer index of chromosome2, the four next highest loadings were
those of the relativelengths of chromosomes 2, 3, 10, and 13. The
second factor,
Table 3. Distribution of centromeric, telomeric, and
peritelomeric heterochromatin in Mantella and Mantidactylusspecies
studied. For each species, we listed numbers of chromosomes on
which a respective band was observed,followed by C and/or D if the
bands stained positively with CMA3 and/or DAPI, respectively.
Chromosome numbers inbrackets refer to faint staining. The
telomeric heterochromatin of M. betsileo was located on the long
arm of the firstchromosome and the short arm of the second
chromosome; the peritelomeric band of M. laevigata was located on
theshort arm of the second chromosome.
Centromeric Telomeric Peritelomericheterochromatin
heterochromatin heterochromatin
Mantella aurantiaca 1–5 (C, D) 1–5 (C) 6, 9, 10, 11 (C,
D)Mantella baroni 1–13 – 6Mantella betsileo 1–13 (D) 1,2 (C)
–Mantella cf. betsileo 1–13 (C, D) 1 (D) –Mantella cowani 1–3 (C)
1–13 (C), 4 (D) –Mantella expectata 1–9, 12–13 (D) 6, 10, 11 (C)
–Mantella laevigata 1–9, 12–13 (D) 8–13 (C) 2 (C)Mantella
madagascariensis [1–5] 1, 8, 9 (C) 6, 10, 11 (D)Mantella nigricans
1, 3, 11 (C, D) 1–10, 12–13 (C, D) 11 (C, D)Mantella pulchra
[1–13?] 1–13 (C) 6, 10, 11 (C, D)Mantella viridis 1–5 (D) 6–13 (C)
–Mantidactylus bicalcaratus [6, 12] (C, D) 1–13 (C) 3
(C)Mantidactylus grandisonae 1–13 (C, D) 1–13 (C) –Mantidactylus
cf. punctatus 1–13 (C, D) 1–13 (C) 3 (C)
Fig. 2. Scatterplot of first and second factors of a PCA of
morphometric chromosome data as given in Table 1 and 2 (ci and
rl).
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Chromosome Data for Malagasy Poison Frogs 509
on the other hand, was mainly influenced by the
centromerindices; the five highest loadings were those of the ci
valuesof the chromosomes 9, 4, 13, 8, and 5. In the
correspondingscatterplot, the three species of Mantidactylus
appeared widelyseparated from all Mantella species (Fig. 2). Among
Mantella,the analysis clustered the species of the M. betsileo
groupaway from the remaining species (along factor 1), while
theother Mantella species did not show a clustering pattern
con-sistent with their attribution to species groups.
Heterochromatin staining resulted in a wide array of
banddistribution and staining patterns. Beside the
centromeric,telomeric and peritelomeric heterochromatin bands
summa-rized in Table 3, paracentromeric bands were found in
M.expectata on chromosomes 1, 3 and 5, and in M. laevigata
onchromosomes 1 and 3. These consisted of DAPI-positivebands
bordered by CMA3-positive bands.
In order to use the karyological data to assess phyloge-netic
relationships, we defined 15 characters based on theresults
presented above: Maximum parsimony analysis of thedata summarized
in Table 4 (using the three Mantidactylusspecies as outgroups; all
characters unordered) failed toresolve relationships among Mantella
species. A strict con-sensus of the most parsimonious cladograms
resulted in abasal polytomy in which Mantidactylus grandisonae
clusteredtogether with the ingroup species. In the
Neighbor-joininganalysis (Fig. 6), bootstrap support >50% was
found for thefollowing groupings: a clade containing Mantella
laevigataand M. expectata, the sister group relationship of this
cladeto M. viridis, and a clade containing M. pulchra and
M.madagascariensis.
DISCUSSION
The results largely support previous data on Mantellakaryology
(Blommers-Schlösser, 1978; Pintak et al., 1998).Generally,
chromosome morphology in Mantella is rather uni-form (all species
have 2n = 26, with five larger and eight smallerchromosome pairs).
However, several of the interspecific dif-ferences observed may
bear taxonomic relevance.
The presence of an acrocentric chromosome pair inMantella
nigricans, unique in the genus, supports the hypoth-esis (Vences et
al., 1999) that this taxon stands on its own atthe species level.
The differentiation found between Mantellabetsileo and M. cf.
betsileo regarding the morphology of thesixth chromosome
(metacentric vs. submetacentric) and theimportant differences in
heterochromatin distribution supportthe hypothesis that these two
forms may be not conspecific.The affinities of the species of the
M. betsileo group to eachother and to M. laevigata is supported by
their general chro-mosome morphology (Fig. 2).
Heterochromatin distribution is quite different amongMantella
species. Actually, at least faint differences wereobserved between
each species included in our study. Hence,the results have to be
interpreted with some caution, as westudied only a limited number
of specimens of each species,and too few data are available on
differentiation between con-specific populations of Mantella.
However, as we studied bothmales and females of most species (see
appendix), we canexclude at least that the differences described
here are erro-neous interpretations of sexual dimorphism.
According to our analyses of Mantella, the same hetero-chromatic
band can be DAPI-positive (thus rich in A+T) or
Table 4. Karyological character states used for phylogenetic
analysis: (1) Configuration of 6th chromosome. 0 submetacentric; 1
metacentric.(2). Configuration of 11th chromosome. 0
submetacentric; 1 acrocentric; 2 metacentric. (3) Intensity of
centromeric C-bands. 0 distinct; 1 faint orvery faint. (4) Presence
of centromeric C-bands. 0 present on all chromosomes; 1 present on
all chromosomes except 10th and 11th; 2 presenton chromosomes 1–5;
3 present on chromosomes 1-3; 4 present on chromosomes 1, 3, 11; 5
present on chromosomes 6, 12. (5) DAPI-stainingof centromeric
C-bands. 0 DAPI-negative; 1 DAPI-positive. (6) CMA-staining of
centromeric C-bands. 0 CMA-negative; 1 CMA-positive. (7)Presence of
telomeric C-bands. 0 absent; 1 scattered on only a few (up to
three) chromosomes; 2 mainly present on chromosomes 1–5; 3
mainlypresent on chromosomes 6–8; 4 present on all or almost all
chromosomes. (8) DAPI-staining of telomeric chromosomes. 0
DAPI-negative; 1DAPI-positive. (9) CMA-staining of telomeric
chromosomes. 0 CMA-negative; 1 CMA-positive. (10) Large
peritelomeric or telomeric C-bandpresent on long arm of 6th
chromosome. 0 absent; 1 present. (11) Large peritelomeric C-bands
on 10th chromosome. 0 absent; 1 present. (12)Large peritelomeric
C-band on 11th chromosome. 0 absent; 1 present. (13)
Paracentromeric bands on 1st and 3rd chromosome. 0 absent;
1present. (14) NOR localization. 0 interstitial on short arm of the
1st chromosome pair; 1 on short of the 2nd chromosome pair. (15)
Peritelomericband on the long arm of the 3rd chromosome pair: 0
absent; 1 present.
Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mantella aurantiaca 0 0 0 2 1 1 2 0 1 1 1 1 0 1 0M. baroni 0 0 1
0 0 0 0 0 0 1 0 0 0 1 0M. betsileo 1 0 0 0 1 0 1 0 1 0 0 0 0 1 0M.
cf. betsileo 0 0 0 0 1 1 1 1 0 0 0 0 0 1 0M. cowani 0 0 0 3 0 1 4
0/1 1 0 0 0 0 1 0M. expectata 0 0 0 1 1 0 1 0 1 0 0 0 1 1 0M.
laevigata 0 0 0 1 1 0 3 0 1 0 0 0 1 1 0M. nigricans 0 1 0 4 1 1 4 1
1 0 0 1 0 1 0M. madagascariensis 0 0 1 2 0 0 1 0 1 1 1 1 0 1 0M.
pulchra 0 0 1 ? 0 0 4 0 1 1 1 1 0 1 0M. viridis 0 0 0 2 1 0 3 0 1 0
0 0 0 1 0Mantidactylus bicalcaratus 1 0 1 5 1 1 4 0 1 0 0 0 0 0 1M.
grandisonae 0 0 0 0 1 1 4 0 1 0 0 0 0 1 0M. cf. punctatus 1 0 0 0 1
1 4 0 1 0 0 0 0 0 1
-
G. Odierna et al.510
CMA3-positive (thus rich in G+C). So far, in different plant
andanimal species, the positive staining of a heterochromatin
bandhas been observed to be limited to either DAPI or CMA3
(John,1988; Schmid and Guttenbach, 1988). Molecular studies ofthe
organization of sex-linked satellite DNA in chicken W chro-
Fig. 3. C-banded karyotype of the 11 Mantella taxa studied: M.
aurantiaca (A), M. baroni (B), M. betsileo (C), M. cf. betsileo
(D), M. cowani (E),M. expectata (F), M. laevigata (G), M.
madagascariensis (H), M. nigricans (I), M. pulchra (J), M. viridis
(K) .
mosomes has shown that specific satellite families are
includedin different chromomeres on the W lampbrush
chromosome(Solovei et al., 1998). The arrangement in different
chro-momeres is a known character in heterochromatic bands(Okada
and Comings, 1974). We therefore hypothesize the
-
Chromosome Data for Malagasy Poison Frogs 511
Fig. 4. C-banded matephase plates of M. pulchra (a and a’), M.
aurantiaca (b and b’), M. madagascariensis (c and c’), M. nigricans
(d and d’),M. baroni (e and e’), M. cowani (f and f’), M. betsileo
(g and g’); M. cf. betsileo (h and h’), M. expectata (i and i’), M.
viridis (j and j’) and M.laevigata (k and k’) successively stained
with Chromomycin A3 ( simple cases) and DAPI ( marked cases).
-
G. Odierna et al.512
Fig. 5. AgNO3 stained metaphase plates (left row), and the same
metaphase plates successively stained with Chromomycin A3 (medium
row)and DAPI ( right row) of Mantidactylus bicalcaratus (a, b, and
c), M. punctatus (d, e, and f) and M. grandisonae (g, h, and i).
The arrows point toNORs.
presence of different families of highly repeated DNAsequences
(one rich in A+T and another one in G+C) inMantella, which are each
arranged in distinct chromomereunits. In different species,
selective amplification of these unitsmay lead (1) to bands
containing either mainly A+T richchromomere units or G+C rich
chromomere units (andthus staining either DAPI+ or CMA+, as in M.
betsileo, M.madagascariensis, and M. viridis); (2) to bands
containing bothtypes of units in comparable proportion (and thus
stainingDAPI+ and CMA+ as in M. aurantiaca, M. cf. betsileo, and
M.pulchra). A similar situation was observed in other
Malagasyanuran genera belonging to the superfamily
Ranoidea(Mantidactylus, Boophis, Heterixalus), and may therefore
bewidespread among ranoid anurans (Odierna, pers. obs.).
Taxonomic and limited phylogenetic relevance withinMantella may
be attributed to the existence of specific loca-tions in which the
accumulation of heterochromatic materialis possible or excluded.
Indeed, within Mantella, two maingroups of species can be
distinguished regarding the hetero-chromatin distribution. In one,
heterochromatin was mainlyfound in the telomeric or peritelomeric
regions of the chromo-somes 6-13 and was largely absent or scarce
in the centro-meric regions of these elements (M. aurantiaca, M.
baroni, M.cowani, M. madagascariensis, M. nigricans, M. pulchra,
andM. viridis). In the other, the centromeric regions of the
chro-
mosomes 6-13 were richer in heterochromatin than theirtelomeric
and peritelomeric regions (M. betsileo, M. cf. betsileo,M.
expectata, and M. laevigata).
Several further groupings are possible based on the
het-erochromatin distribution patterns. Mantella aurantiaca,
M.madagascariensis, and M. pulchra share the presence of dis-tinct
peritelomeric bands on the 6th, 10th, and 11th chromo-some.
Mantella aurantiaca additionally has a peritelomericband on the 9th
chromosome. This largely corresponds to thedata of Pintak et al.
(1998) who have found peritelomeric bandson the 6th, 11th, and 12th
chromosome in M. aurantiaca (cor-rect ordering of the small
chromosomes is often difficult andmay vary according to the method
used). Pintak et al. (1998)have further observed peritelomeric
bands on the 6th and 11thchromosome of M. crocea, indicating that
it is also closelyrelated to M. aurantiaca, M. madagascariensis,
and M. pulchra.This is also corroborated by allozyme data which
stronglysupport a monophyletic group containing these four
species(Vences et al., 1998c). A peritelomeric C-band on the 6th
chro-mosome also occurred in M. baroni, as indicated by the
presentdata and data in Pintak et al. (1998). In contrast,
allozymedata and osteology clearly indicate that M. baroni is part
of amonophyletic group containing also M. cowani and M.
nigricans(Vences et al., 1998b,c). Thus, the band on the 6th
chromo-some may be not homologous in M. baroni and in the M.
-
Chromosome Data for Malagasy Poison Frogs 513
madagascariensis and M. aurantiaca groups (as also sup-ported by
the different reaction to CMA3 and DAPI stainingsof the band in the
three groups). Alternatively, the band mayhave been lost in other
members of the M. baroni group.
Another species pair with similar heterochromatin distri-bution
comprised M. laevigata and M. expectata. These arethe only species
which showed adjacent separate bands inpericentromeric areas which
were either CMA+ or DAPI+.Mantella expectata belongs to the M.
betsileo group whichalso contains M. viridis, whereas M. laevigata
has an isolatedposition within the genus (Vences et al., 1998c,
1999). BothM. laevigata and the M. betsileo group are thought to be
basalgroups within Mantella (Vences et al., 1998b,c), but a
sister-group relationship of M. laevigata and M. expectata is
contra-dicted by allozyme data (Vences et al., 1998c).
The location of the NOR has been demonstrated to be
ofphylogenetic and taxonomic validity in many animal
groupsincluding amphibians, reptiles, and fish (Amemiya andGold,
1990; King, 1990; Olmo et al., 1993). In the genusMantidactylus,
Aprea et al. (1998) have found variability ofthe NOR location among
different species groups and sub-genera but, on the other hand, a
constant state within thesegroups. The state occurring in Mantella
is similar toMantidactylus grandisonae (subgenus Blommersia)
followingthe data presented herein. However, it differs from the
statesin the subgenera Brygoomantis (Mantidactylus alutus, Apreaet
al., 1998) Gephyromantis (Mantidactylus luteus, Aprea etal., 1998;
M. silvanus, Odierna, unpubl.), Pandanusicola
(Mantidactylus bicalcaratus, M. cf. punctatus, data herein),and
Phylacomantis (Mantidactylus redimitus, Aprea et al.,1998). Vences
et al. (1998b) have hypothesized that the sub-genera Guibemantis,
Blommersia, and Pandanusicola maybe the closest extant relatives to
Mantella. The chromosomaldata indicate that Blommersia probably is
a better candidatefor the sister group of Mantella than
Pandanusicola. No NORdata are available so far for Guibemantis.
The analysis of intrageneric Mantella relationships basedon
karyological characters did not provide adequate phyloge-netic
resolution (Fig. 6). The karyological characters studiedmay have
evolved either too slow (NOR) or too fast (hetero-chromatin
distribution) to match the main cladogenetic eventswithin Mantella.
The NOR data appear to be more informativefor rather old splits
(e.g., the separation of the Blommersia-Mantella clade from other
Mantidactylus) whereas the het-erochromatin is so quickly
re-distributed that only presumablyvery young groups (such as the
clade containing Mantellaaurantiaca, M. madagascariensis, and M.
pulchra which aregenetically extremely similar according to Vences
et al., 1998c)conserve some common, slightly informative
patterns.
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(Received October 23, 2000 / Accepted January 29, 2001)
APPENDIX: MATERIAL EXAMINED
Material without locality data was obtained through the pet
trade; some specimens examined were destroyed for the analysis and
thus notpreserved, accounting for minor discrepancies between
number of examined and catalogued specimens.
Mantella aurantiaca, one male and four females, ZFMK
72001–72004, 72143; M. baroni, two males and two juveniles, ZFMK
72008-72009,72146; M. betsileo, two males and three females, ZFMK
72017–72020, 72002; one male and one female, ZFMK 72017 and 72020,
captive-bredfrom a stock from Nosy Be, NW-Madagascar; M. cf.
betsileo, one female, ZFMK 72024, from near Morondava, W
Madagascar; M. cowani, twomales, ZFMK 72014, 72149; M. expectata,
one male and two juveniles, ZFMK 72147, 72021, 72023; M. laevigata,
three males and one juvenile,ZFMK 72010–72013; M. madagascariensis,
one male, one female, and two juveniles, ZFMK 72005–72007, 72148;
M. nigricans, one male,ZFMK 72015; M. pulchra, one male and one
female, ZFMK 72142; M. viridis, one male and one female, ZFMK
72016, 72145; Mantidactylusbicalcaratus, one male and two females,
MRSN A1977.1, from Ambolokopatrika Rainforest (between
Anjanaharibe-Sud and Marojejy),Andranomadio (campsite 2), 14°32 S,
49°26’ E, 860 m; M. grandisonae, one male, MRSN A1975.1 (FN 7806),
from Foret de Beanjada, MasoalaNational Park, 15°17’ S, 49°60’ E,
620 m; M. cf. punctatus, two females, MRSN A1976.1, Ambolokopatrika
Rainforest (between Anjanaharibe-Sud and Marojejy), Andranomadio
(campsite 2), 14°32’ S, 49°26’ E, 860 m.